This meeting was organized to review scientific progress in research on recombinant DNA molecules and to discuss appropriate
ways to deal with the potential biohazards of this work. Impressive scientific achievements have already been made in this
field and these techniques have a remarkable potential for furthering our understanding of fundamental biochemical processes
in pro- and eukaryotic cells. The use of recombinant DNA methodology promises to revolutionize the practice of molecular
biology. While there has as yet been no practical application of the new techniques, there is every reason to believe that
they will have significant practical utility in the future.

Of particular concern to the participants at the meeting was the issue of whether the pause in certain aspects of research
in this area, called for by the Committee on Recombinant DNA Molecules of the National Academy of Sciences, U.S.A. in the
letter published in July, 1974, should end; and, if so, how the scientific work could be undertaken with minimal risks to
workers in laboratories, to the public at large and to the animal and plant species sharing our ecosystems.

The new techniques, which permit combination of genetic information from very different organisms, place us in an area of
biology with many unknowns. Even in the present, more limited conduct of research in this field, the evaluation of potential
biohazards has proved to be extremely difficult. It is this ignorance that has compelled us to conclude that it would be
wise to exercise considerable caution in performing this research. Nevertheless, the participants at the Conference agreed
that most of the work on construction of recombinant DNA molecules should proceed provided that appropriate safeguards, principally
biological and physical barriers adequate to contain the newly created organisms , are employed. Moreover, the standards
of protection should be greater at the beginning and modified as improvements in the methodology occur and assessments of
the risks change. Furthermore, it was agreed that there are certain experiments in which the potential risks are of such
a serious nature that they ought not to be done with presently available containment facilities. In the longer term serious
problems may arise in the large scale application of this methodology
in industry, medicine and agriculture. But it was also recognized that future research and experience may show that many
of the potential biohazards are less serious and/or less probable than we now suspect.

II. Principles Guiding the Recommendations and Conclusions

Though our assessments of the risks involved with each of the various lines of research on recombinant DNA molecules may differ,
few, if any, believe that this methodology is free from any risk. Reasonable principles for dealing with these potential risks
are: 1) that containment be made an essential consideration in the
experimental design and, 2) that the effectiveness of the containment should match, as closely as possible, the estimated
risk. Consequently, whatever scale of risks is agreed upon, there should be a commensurate scale of containment. Estimating
the risks will be difficult and intuitive at first but this will improve as we acquire additional knowledge; at each stage
we shall have to match the potential risk with an appropriate level of containment. Experiments requiring large scale operations
would seem to be riskier than equivalent experiments done on a small scale and, therefore, require more stringent containment
procedures. The use of cloning vehicles or vectors (plasmids, phages) and bacterial hosts with a restricted
capacity to multiply outside of the laboratory would reduce the potential biohazard of a particular experiment. Thus, the
ways in which potential biohazards and different levels of containment are matched may vary from time to time particularly
as the containment technology is improved. The means for assessing and balancing risks with appropriate levels of containment
will need to be reexamined
from time to time. Hopefully, through both formal and informal channels of information within and between the nations of the
world, the way in which potential biohazards and levels of containment are matched would be consistent.

Containment of potentially biohazardous agents can be achieved in several ways. The most significant contribution to limiting
the spread of the recombinant DNAs, is the use of biological barriers. These barriers are of two types: 1) fastidious bacterial
hosts unable to survive in natural environments, and 2) non-transmissible and equally fastidious vectors (plasmids, bacteriophages
or other viruses) able to grow only in specified hosts. Physical containment, exemplified by the use of suitable hoods, or
where applicable, limited access or negative pressure laboratories, provides an additional factor of safety. Particularly
important is strict adherence to good microbiological practices which, to a large measure can limit the escape of organisms
from the experimental situation, and thereby increase the safety of the operation. Consequently, education and training of
all personnel involved in the experiments is essential to the effectiveness of all containment measures. In practice these
different means of containment will complement one another and documented substantial improvements in the ability to restrict
the growth of bacterial hosts and vectors could permit modifications of the complementary physical containment requirements.

Stringent physical containment and rigorous laboratory procedures can reduce but not eliminate the possibility of spreading
potentially hazardous agents. Therefore, investigators relying upon "disarmed" hosts and vectors for additional safety
must rigorously test the effectiveness of these agents before accepting their validity as
biological barriers.

III. Specific Recommendations for Matching Types of Containment with Types
of Experiments

No classification of experiments as to risk and no set of containment procedures can anticipate all situations. Given our
present uncertain ties about the hazards, the parameters proposed here are broadly conceived and meant to provide provisional
guidelines for investigators and agencies concerned with research on recombinant DNAs. However, each investigator bears a
responsibility for determining whether, in his particular case, special circumstances warrant a higher level of containment
than is suggested here.

A. Types of Containment

1. Minimal Risk: This type of containment is intended for experiments in which the biohazards may be accurately assessed and
are expected to be minimal. Such containment can be achieved by following the operating procedures recommended for clinical
microbiological laboratories. Essential features of such facilities are no drinking, eating or smoking in the laboratory,
wearing laboratory
coats in the work area, the use of cotton-plugged pipettes or prefer ably mechanical pipetting devices and prompt disinfection
of contaminated materials.

2. Low Risk: This level of containment is appropriate for experiments which generate novel biotypes but where the available
information indicates that the recombinant DNA cannot alter appreciably the ecological behavior of the recipient species,
increase significantly its pathogenicity, or prevent effective treatment of any resulting infections. The key features of
this containment (in addition to the
minimal procedures mentioned above) are a prohibition on mouth pipetting, access limited to laboratory personnel, and the
use of biological safety cabinets for procedures likely to produce aerosols (e.g., blending and sonication). Though existing
vectors may be used in conjunction with low risk procedures, safer vectors and hosts should be adopted as they become available.

3. Moderate Risk: Such containment facilities are intended for experiments in which there is a probability of generating an
agent with a significant potential for pathogenicity or ecological disruption. The principle features of this level of containment,
in addition to those of the two preceding classes, are that transfer operations should be carried out in biological safety
cabinets (e.g., laminar flow hoods), gloves should be worn during the handling of infectious materials, vacuum lines must
be protected by filters and negative pressure should be maintained in the
limited access laboratories. Moreover, experiments posing a moderate risk must be done only with vectors and hosts that have
an appreciably impaired
capacity to multiply outside of the laboratory.

4. High Risk: This level of containment is intended for experiments in which the potential for ecological disruption or pathogenicity
of the modified organism could be severe and thereby pose a serious biohazard to laboratory personnel or the public. The main
features of this type of facility, which was designed to contain highly infectious microbiological agents, are its isolation
from other areas by air locks, a negative pressure environment, a requirement for clothing changes and showers for entering
personnel and laboratories fitted with treatment systems to inactivate or remove biological agents that may be contaminants
in exhaust air, liquid and solid wastes. All persons occupying these areas should wear protective
laboratory clothing and shower at each exit from the containment facility. The handling of agents should be confined to biological
safety cabinets in which the exhaust air is incinerated or passed through Hepa filters. High risk containment includes, beside
the physical and procedural features described above, the use of rigorously tested vectors and hosts whose growth can be confined
to the laboratory.

B. Types of Experiments

Accurate estimates of the risks associated with different types of experiments are difficult to obtain because of our ignorance
of the probability that the anticipated dangers will manifest themselves. Nevertheless, experiments involving the construction
and propagation of recombinant DNA molecules using DNAs from 1) prokaryotes, bacteriophages and other plasmids, 2) animal
viruses, and 3) eukaryotes have been characterized as minimal, low, moderate and high risks to
guide investigators in their choice of the appropriate containment. These designations should be viewed as interim assignments
which will need to be revised upward or downward in the light of future experience.

The recombinant DNA molecules themselves, as distinct from cells carrying them, may be infectious to bacteria or higher organisms.
DNA preparations from these experiments, particularly in large quantities, should be chemically inactivated before disposal.

1. Prokaryotes, bacteriophages and bacterial plasmids:

Where the construction of recombinant DNA molecules and their propagation involves prokaryotic agents that are known to exchange
genetic information naturally, the experiments can be performed in minimal risk containment facilities. Where such experiments
pose a potential hazard, more stringent containment may be warranted.

Experiments involving the creation and propagation of recombinant DNA molecules from DNAs of species that ordinarily do not
exchange genetic information, generate novel biotypes. Because such experiments may pose biohazards greater than those associated
with the original organisms, they should be performed, at least, in low risk containment facilities. If the experiments involve
either pathogenic organisms, or genetic determinants that may increase the pathogenicity of the recipient species, or if the
transferred DNA can confer upon the recipient organisms new metabolic activities not native to these species and thereby modify
its relationship with the environment, then moderate or high risk containment should be used.

Experiments extending the range of resistance of established human pathogens to therapeutically useful antibiotics or disinfectants
should be undertaken only under moderate or high risk containment depending upon the virulence of the organism involved.

2. Animal Viruses: Experiments involving linkage of viral genomes or genome segments to prokaryotic vectors and their propagation
in prokaryotic cells should be performed only with vector-host systems having demonstrably restricted growth capabilities
outside the laboratory and with moderate risk containment facilities. Rigorously purified and characterized segments of non-oncogenic
viral genomes or of the demonstrably non-transforming regions of oncogenic viral DNAs can be attached to presently existing
vectors and propagated in moderate risk containment
facilities; as safer vector-host systems become available such experiments may be performed in low risk facilities.

Experiments designed to introduce or propagate DNA from non-viral or other low risk agents in animal cells should use only
low risk animal DNAs as vectors (e.g., viral , mitochondrial) and manipulations should be confined to moderate risk containment
facilities.

3. Eukaryotes: The risks associated with joining random fragments of eukaryote DNA to prokaryotic DNA vectors and the propagation
of these recombinant DNAs in prokaryotic hosts are the most difficult to assess.

A priori, the DNA from warm-blooded vertebrates is more likely to contain cryptic viral genomes potentially pathogenic for
many than is the DNA from other eukaryotes. Consequently, attempts to clone segments of DNA from such animal and particularly
primate genomes should be performed only with vector-host systems having demonstrably restricted growth capabilities outside
the laboratory and in a moderate risk containment facility. Until cloned segments of warm-blooded vertebrate DNA are completely
characterized, they should continue to be maintained in the most restricted vector-host system in moderate risk containment
laboratories; when such cloned segments are characterized, they may be
propagated as suggested above for purified segments of virus genomes.

Unless the organism makes a product known to be dangerous (e.g., toxin, virus), recombinant DNAs from cold-blooded vertebrates
and all other lower
eukaryotes can be constructed and propagated with the safest vector-host
system available in low risk containment facilities.

Purified DNA from any source that performs known functions and can be judged to be non-toxic, may be cloned with currently
available vectors in low risk containment facilities. (Toxic here includes potentially oncogenic products or substances that
might perturb normal metabolism if produced in an animal or plant by a resident microorganism.)

4. Experiments to be Deferred: There are feasible experiment which present such serious dangers that their performance should
not be undertaken at this time with the currently available vector-host systems and the presently available containment capability.
These include the cloning of recombinant DNAs derived from highly pathogenic organisms (i.e., Class 111, IV, V etiologic agents
as classified by the United Stated Department of Health, Education and Welfare), DNA containing toxin genes and large scale
experiments (more than 10 liters
of culture) using recombinant DNAs that are able to make products potentially harmful to man, animals or plants.

IV. Implementation

In many countries steps are already being taken by national bodies to formulate codes of practice for the conduct of experiments
with known or potential biohazard.+* Until these are established, we urge individual scientists to use the proposals in this
document as a guide. In addition, there are some recommendations which could be immediately and directly implemented by the
scientific community.

A. Development of Safer Vectors and Hosts

An important and encouraging accomplishment of the meeting was the realization that special bacteria and vectors can be constructed
genetically, which have a restricted capacity to multiply outside the laboratory, and

*Advisory Board for the Research Councils. Report of the Working Party on the Experimental Manipulation of the Genetic Composition
of Micro-Organisms.
Presented to Parliament by the Secretary of State for Education and Science by Command of Her Majesty January 1975. London:
Her Majesty's Stationery
Office, 1975, 23pp.

that the use of these organisms could enhance the safety of recombinant DNA experiments by many orders of magnitude. Experiments
along these lines are presently in progress and in the near future, variants of A bacteriophage, non-transmissible plasmids
and special strains of E. coli will become available. All of these vectors could reduce the potential biohazards by very large
factors and improve the methodology as well. Other vector-host systems, particularly modified strains of Bacillus subtilis
and their relevant bacteriophages and plasmids, may also be useful for particular purposes. Quite possibly safe and suitable
vectors may be found for eukaryotic hosts such as yeast and readily cultured plant and animal cells. There is likely to be
a continuous development in this area and the participants at the meeting agreed that improved vector-host systems which reduce
the biohazards of recombinant DNA research will be made freely available to all interested investigators.

B. Laboratory Procedures

It is the clear responsibility of the principal investigator to inform the staff of the laboratory of the potential hazards
of such experiments, before they are initiated. Free and open discussion is necessary so that each individual participating
in the experiment fully understands the nature of the experiment and any risk that might be involved. All workers must be
properly trained in the containment procedures that are designed to control the hazard, including emergency actions in the
event of a hazard. It is also recommended that appropriate health surveillance of all personnel, including serological monitoring,
be conducted periodically.

C. Education and Reassessment

Research in this area will develop very quickly and the methods will be applied to many different biological problems. At
any given time it is impossible to foresee the entire range of all potential experiments and make judgments on them. Therefore,
it is essential to undertake a continuing reassessment of the problems in the light of new scientific knowledge. This could
be achieved by a series of annual workshops and meetings, some of which should be at the international level. There should
also be courses to train individuals in the relevant methods since it is likely that the work will be taken up by laboratories
which may not have had
extensive experience in this area. High priority should also be given to research that could improve and evaluate the containment
effectiveness of new and existing vector-host systems.

V. New Knowledge

This document represents our first assessment of the potential biohazards in the light of current knowledge. However, little
is known about the survival of laboratory strains of bacteria and bacteriophages in different ecological niches in the outside
world. Even less is known about whether recombinant DNA molecules will enhance or depress the survival of their vectors and
hosts in nature. These questions are fundamental to the testing of any new organism that may be constructed. Research in this
area needs to be undertaken and should be given high priority. In general, however, molecular biologists who may construct
DNA recombinant molecules do not undertake these experiments and it will be necessary to facilitate collaborative research
between them and groups skilled in the study of bacterial infection or ecological microbiology. Work should also be undertaken
which would enable us to monitor the escape or dissemination of cloning vehicles and their hosts.

Nothing is known about the potential infectivity in higher organisms of phages or bacteria containing segments of eukaryotic
DNA and very little about the infectivity of the DNA molecules themselves. Genetic transformation of bacteria does occur in
animals suggesting that recombinant DNA molecules can retain their biological potency in this environment. There are many
questions in this area, the answers to which are essential for our assessment of the biohazards of experiments with recombinant
DNA molecules. It will be necessary to ensure that this work will be planned and carried out; and it will be particularly
important to have this information before large scale applications of the use of recombinant DNA molecules is attempted.